Legionella pneumophila (Lp), the etiologic agent of Legionnaires' disease is an intracellular pathogen of mononuclear phagocytic cells and of several species of fresh water protozoa including the amoebae Hartmannella verminformis (Hv). Since the discovery of the bacterial-protozoal interaction, there has been much speculation, but limited research, directed towards elucidating the relevance of this natural parasitism to human infection. Using a murine model of Legionnaires' disease, we have demonstrated that L. pneumophila-infected H. verminformis (Lp-infected Hv) are an infectious particle in replicative Lp lung infection and are more pathogenic than Lp alone. The objective of this proposal is to identify specific mechanisms responsible for enhance pathogenicity of Lp-infected Hv. Our central hypothesis is that intrapulmonary Hv provide a nice for LP replication, enhance Lp virulence, and/or alter host immune responses required for resolution of replicative Lp lung infection. To test this hypothesis, we will 1) identify co-culture conditions which maximize the pathogenicity of Lp infected Hv; 2) use Lp mutants avirulent for amoebae to determine if Hv are a niche for Lp replication; 3) compare adherence, invasion, intracellular trafficking and replication of Lp grown in amoebae and broth; 4) compare immune responses elicited by Lp-infected Hb and Lp. Methods: Lp-infected Hv, prepared at different stages of growth and multiplicities of infection, will be administered intratracheally and by aerosol to A/J mice. Conditions which maximize the pathogenicity of Lp- infected Hv will be identified. The role of intrapulmonary Hv as a niche for Lp replication will be identified by co-inoculating A/J mice with Hv and mutant strains of Lp which are selectively avirulent for Hv. Effects of growth of Lp in Hv on bacterial virulence, phenotypic (adherence, invasion, intracellular growth and trafficking) and competency to produce disease will be assessed. Effects of Lp-infected Hv on induction of specific cytokines which facilitate resolution of Lp lung infection and/or cytokine-mediated lung injury (gamma interferon, tumor necrosis factor alpha, interleukin-1) and their natural inhibitors (the TNF soluble receptor, IL-1 receptor antagonist and interleukin-10) will be determined. Significance: The proposed studies will identify specific mechanisms by which Lp-infected Hv enhance the severity of replicative Lp lung infection. Results of these studies will facilitate the design of new strategies to prevent and/or treat legionellosis. Furthermore, this model system may identify mechanisms by which inhaled protozoa augment lung diseases induced by inhaled microbes.